Acute lymphoblastic leukemia (ALL) is an aggressive immature lymphoid neoplasm. Over the last couple of decades, there has been substantial improvement in disease outcomes for these patients [3]. In part, this improvement is due to enhanced identification of patients requiring additional therapy based on the assessment of minimal residual disease (MRD). Currently, assessment of MRD is achieved through the use of either patient-specific molecular assays (predominantly in Europe), and/or multi-parametric flow cytometry (mpFC), (commonly in the US). At present, implementation of these approaches in a uniform manner is complex and challenging. Individualized molecular assessment of MRD is limited by requirements for a large institutional infrastructure, as molecular assays for each patient must be individually-designed and validated. By contrast, multi-parametric flow cytometry is difficult to standardize, resulting in disparate quality. While it is clear that MRD i important for guiding patient-specific care, current approaches are not robust to achieve this goal consistently. In a preliminary project involving 43 paired pre- and post-treatment samples from an ongoing T-ALL Children's Oncology Group (COG) trial AALL0434 [1], we demonstrate that high-throughput sequencing of T-cell receptor gene rearrangements enhanced detection of very low-level MRD, improving upon the sensitivity and specificity of multi-parametric flow cytometry, without requiring the complex institutional infrastructure required to individualize high-sensitivity molecular evaluation for minimal residual disease. We found that high-throughput sequencing could identify MRD in all patients in whom multi-parametric flow cytometry identified disease. However, sequencing also permitted detection of MRD at a higher sensitivity of approximately 10-fold. We have since completed analysis of another cohort of 99 patients with B-lineage acute lymphoblastic leukemia derived from COG trial AALL0932 (manuscript in preparation). In this study, we also found that next-generation sequencing of immunoglobulin receptor gene rearrangements could provide enhanced detection of MRD. Several questions are raised by these data. First, what is the clinical specificity and sensitivityof these findings? Second, to what extent is next-generation sequencing suitable for routine implementation in the clinical laboratory? In this proposal, we apply high-throughput sequencing to sensitively and comprehensively assess MRD in patients with B- and T-lineage ALL.